d(GC) - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 6 (2) 229-240 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)
Table 4. An L 9 Taguchi array for IFNs PEGylation.
Trial Polymer Protein/mPEG HPLC Ninhydrin
Number MW (kDa) pH fractions Response Response
1 5.0 7.4 1:10 0.879 0.128
2 12.5 7.7 1:10 0.838 0.179
3 20.0 8.0 1:10 0.805 0.218
4 5.0 7.7 1:25 0.774 0.256
5 12.5 8.0 1:25 0.712 0.271
6 20.0 7.4 1:25 0.621 0.280
7 5.0 8.0 1:40 0.605 0.412
8 12.5 7.4 1:40 0.611 0.390
9 20.0 7.7 1:40 0.579 0.431
the result is represented as the ratio of the
absorption difference between the non-
PEGylated and PEGylated to the absorption of
non-PEGylated protein. Therefore, in Ninhydrin
test, a higher ratio shows higher levels of polymer
attachment to the protein.
Full factoriel design: By considering the
obtained results by HPLC and Ninhydrin tests,
full factorial design was employed to screen the
main effective factors on PEGylation, using linear
polymers with molecular weights of 5 and 20 kDa.
Table 2 shows data regarding the full factorial
design using linear PEGs of 5 and 20 kDa.
According to Table 2, by increasing the level of
pH, polymer molecular weight, and the ratio of
protein to mPEG fractions, HPLC responses
have decreased and Ninhydrin responses
increased, indicating that the degree of PEG
attachment to the protein has increased. Diwan
and Park reported that by increasing the polymer
molecular weight, pH, and the ratio of protein to
mPEG fractions during PEGylation of IFN-β with
mPEG-SPA, the degree of PEGylation increased
(28). By considering the analysis of variance
(ANOVA) and p-values (Table 5) less than 0.01,
the most important factors were determined.
These results show that PEG molecular weight
(A), pH (B), and the ratio of protein to mPEG
fractions (C) are important but the effect of their
interactions are not significant.
Ahmad Abolhasani et al
234
Taguchi statistical design: By considering the
important factors, Taguchi method (L 9 array) was
used to obtain the optimum conditions using the
linear PEGs. Table 4, shows the obtained results
by Taguchi design using linear PEGs of 5 and 20
kDa. According to Table 4, by increasing the
levels of pH, polymer molecular weight and the
ratio of protein to mPEG fractions, HPLC
responses have decreased and Ninhydrin
responses increased, indicating an increase in
attachment of PEG to the protein. This shows
that the amount of protein PEGylation has been
improved by increasing the level of the factors.
Hence the optimum conditions for PEGylation
that were obtained by software are as follows:
pH 8; the ratio of protein to mPEG fractions 1:40;
PEG molecular weight 20 kDa.
One factor at a time design: Figure 2 shows
the obtained results for one factor at a time design
using 40 kDa branched PEG. PEG attachment
to the protein increased following increases in
pH and the ratio of mPEG to protein, as HPLC
responses decreased and Ninhydrin responses
increased. It shows the optimum condition for
PEGylation reaction using the branched 40 kDa
PEG, as pH 8 and the ratio of protein to mPEG
fractions 1:40. At this point, the non PEGylated
protein component is at its minimum and the
numbers of the reacted amino groups are at their
maximum. Table 6 shows the optimum conditions